Inflammatory bowel disease is characterized by a chronic
inflammation of the intestinal mucosa. The mucosal epithelium of the
alimentary tract constitutes a key element of the mucosal barrier to a
broad spectrum of deleterious substances present within the intestinal
lumen including bacterial microorganisms, various dietary factors,
gastrointestinal secretory products and drugs. In addition, this mucosal
barrier can be disturbed in the course of various intestinal disorders
including inflammatory bowel diseases. Fortunately, the integrity of the
gastrointestinal surface epithelium is rapidly reestablished even after
extensive destruction. Rapid resealing of the epithelial barrier
following injuries is accomplished by a process termed epithelial
restitution, followed by more delayed mechanisms of epithelial wound
healing including increased epithelial cell proliferation and epithelial
cell differentiation. Restitution of the intestinal surface epithelium
is modulated by a range of highly divergent factors among them a broad
spectrum of structurally distinct regulatory peptides, variously
described as growth factors or cytokines. Several regulatory peptide
factors act from the basolateral site of the epithelial surface and
enhance epithelial cell restitution through TGF-b-dependent
pathways. In contrast, members of the trefoil factor family (TFF
peptides) appear to stimulate epithelial restitution in conjunction with
mucin glycoproteins through a TGF-b-independent
mechanism from the apical site of the intestinal epithelium. In
addition, a number of other peptide molecules like extracellular matrix
factors and blood clotting factors and also non-peptide molecules
including phospholipids, short-chain fatty acids (SCFA), adenine
nucleotides, trace elements and pharmacological agents modulate
intestinal epithelial repair mechanisms. Repeated damage and injury of
the intestinal surface are key features of various intestinal disorders
including inflammatory bowel diseases and require constant repair of the
epithelium. Enhancement of intestinal repair mechanisms by regulatory
peptides or other modulatory factors may provide future approaches for
the treatment of diseases that are characterized by injuries of the
epithelial surface.

The surface of the digestive tract is covered by
epithelial cells that constitute an efficient physical barrier between
the dietary and enteric flora pathogens found in the intestinal lumen
and the individuum, but also allows an exchange between nutrients and
the systemic circulation[1]. The epithelial defense mechanism
can be categorized into three key components: pre-epithelial, epithelial
and post-epithelial, the latter is represented by the lamina propria[2].
The pre-epithelial mucus barrier is composed of mucin associated with
other proteins and lipids and forms a continuous gel into which a
bicarbonate-rich fluid is secreted, maintaining a neutralizing pH at the
epithelial surface. Phosphatidylcholine is the predominant surface
bioactive phospholipid found within the gastrointestinal tract[3].
Intestinal epithelial cells secrete mucins and glycocalyx, which contain
membrane-anchored negatively charged mucin-like glycoproteins and
hydrophobic phospholipids[4]. The tight adherence of mucin to
the apical surfaces of epithelia is owed to the existence of the
specific complex between mucin oligosaccharides and the mucin binding
protein of the apical mucosal membrane[5]. The hydrophobic
lining of the luminal surface has an important functional role. It
prevents microorganisms from getting into contact with and to adhering
to the plasma membrane. It furthermore protects the mucosal epithelium
against chemical and mechanical injuries[6]. Epithelial cells
provide the second line of the mucosal defense system. Whereas in the
upper digestive tract this layer consists of a stratified epithelium,
the stomach, small, and large bowel are surfaced with a simple
epithelial layer sealed by tight junctions[7]. When intact,
the uptake of antigens, macro- and microorganism through this layer is
restricted by luminal cell-surface structures. The mucosal surface
epithelial cells are rapidly proliferating with a complete turnover
every 24 to 96 h[8]. The proliferative compartment of
epithelial cells is localized in the crypt region and is segregated from
a gradient of increasingly differentiated epithelial cells present along
the vertical axis of the functional villus compartment[9,10].

INTESTINAL WOUND HEALING

Damage and impairment of the intestinal surface barrier
are observed in the course of various diseases and may result in an
increased penetration and absorption of toxic and immunogenic factors
into the body leading to inflammation, uncontrolled immune response, and
disequilibrium of the homeostasis of the host. Thus, rapid resealing of
the epithelial surface barrier following injuries or physiological
damage is essential to preserve the normal homeostasis. Observations
over the past several years have demonstrated the ability of the
intestinal tract to rapidly reestablish the continuity of the surface
epithelium after extensive destruction[11-14]. The continuity
of the epithelial surface is reestablished by at least three distinct
mechanisms. First, epithelial cells adjacent to the injured surface
migrate into the wound to cover the denuded area. Those epithelial cells
that migrate into the wound defect dedifferentiate, form
pseudopodia-like structures, reorganize their cytoskeleton, and
redifferentiate after closure of the wound defect. This process has been
termed epithelial restitution and does not require cell proliferation[15].
Intestinal epithelial restitution occurs within minutes to hours both
in vivo and in vitro. Secondly, epithelial cell proliferation
is necessary to replenish the decreased cell pool. Third, maturation and
differentiation of undifferentiated epithelial cells is needed to
maintain the numerous functional activities of the mucosal epithelium.
The separation of intestinal epithelial wound healing in three distinct
processes is rather artificial and simplified. These three wound-healing
processes overlap and distinct processes may not be observed in vivo
where these processes overlap. The preservation of this barrier
following injuries is regulated by a broad spectrum of structurally
distinct regulatory factors, including cytokines, growth factors,
adhesion molecules, neuropeptides and phospholipids[16-19].
However, this artificial and simplified model provides a tool to better
understand the physiology and pathophysiology of intestinal epithelial
wound healing. Moreover, deeper lesions or penetrating injuries will
require additional repair mechanisms that involve inflammatory processes
and non-epithelial cell populations. Inflammatory processes especially
may interfere with epithelial cell migration and proliferation and thus
modulate intestinal epithelial healing.

IMPORTANT MODULATORS OF THE INTESTINAL EPITHELIAL CELL
FUNCTION

The epithelial cell populations of the intestinal mucosa
are modulated by a number of factors that are present within the lumen,
the epithelium itself or the underlying lamina propria (Table
1). Although the full variety of regulatory factors that play a
role in the control of intestinal epithelial and non-epithelial cell
populations has not been fully defined yet, there is increasing
appreciation of the diversity of these factors in general and the
importance of several specific peptide and non-peptide factors produced
or released within the intestine. The identification and
characterization of numerous regulatory peptide and non-peptide factors
has led to the recognition of a network of interrelated factors within
the intestine (Figure
1). The constituents of this network generally possess multiple
functional properties and exhibit pleiotropism in their cellular sources
and targets. As a result, this network is highly redundant in several
dimensions[20]. Regulatory peptides especially seem to play a
key role in intestinal epithelial wound repair, as they are abundantly
detectable in the intestinal lumen, intestinal epithelium and the
underlying lamina propria. Various members of several distinct
regulatory peptide families have been recognized to modulate a broad
spectrum of intestinal epithelial cell functions including cell
migration, proliferation and/or differentiation (Figure
2). As outlined above the latter epithelial cell functions are
highly relevant for the modulation of intestinal epithelial wound
repair.

THE ROLE OF REGULATORY PEPTIDES FOR INTESTINAL EPITHELIAL
WOUND HEALING

A broad
spectrum of structurally distinct regulatory peptides is expressed from
various cell populations within the mucosa of the intestinal tract.
These regulatory peptides, conventionally designated as growth factors
and cytokines play an essential role in regulating differential
epithelial cell functions in order to preserve normal homeostasis and
integrity of the intestinal mucosa[20-25]. The terminology of
regulatory peptides is often confusing and arbitrary. The term cytokines
is now increasingly used to describe a bunch of regulatory peptides that
can be variously identified as regulatory peptides, peptide growth
factors, interleukins, interferons and colony stimulating or
hematopoetic stem cell factors. For the purpose of simplification, the
term regulatory peptide or cytokine will be used in this paper to
address all the different classes of regulatory peptide factors.Regulatory peptides can be reasonably classified on the basis of
structural homologies and disparities into several discrete families.
Peptide growth factor families and selected members with functional
activities in the modulation of intestinal wound healing. In addition to
growth factor families, a number of regulatory peptide factors,
seemingly without structural similarities to other regulatory peptide
family members like vascular endothelial cell growth factor (VEGF) and
platelet-derived growth factor (PDGF) have been identified to be
expressed within the intestinal tract and to modulate wound healing
properties within the intestinal mucosa[25]. Furthermore, a
countless number of classical cytokines like IL-1, IL-2, IL-15, IL-22
and IFN-g
are expressed within the intestine and modulate numerous intestinal
epithelial cell functions[26-30].

The various
effects of a number of regulatory peptides on cell adhesion, migration,
proliferation, differentiation, intestinal epithelial barrier function
and angiogenesis suggest that these peptides are likely relevant factors
for intestinal repair mechanisms. Both in-vitro and in-vivo
studies have demonstrated that several growth factors and cytokines
can enhance epithelial cell restitution[31,32]. A spectrum of
growth factors and cytokines including EGF, VEGF, HGF, GLP-2, various
FGF peptides, IL-1, IL-2 and IFN-g
have been demonstrated to enhance epithelial cell restitution through a
TGF-b-dependent
pathway[33-36]. Interestingly, it appears that
restitution-enhancing cytokines use different mechanisms to modulate TGF-b-peptide
levels. While TGF-b,
EGF, IL-1, IFN-g
and HGF only increased the concentration of bioactive TGF-b,
acidic and basic FGF and also IL-2 enhanced both the bioactivation of
TGF-b
and the expression of TGF-b
mRNA and production of latent TGF-b
peptide. This might reflect a different mechanism, by which acidic and
basic FGF and also IL-2 modulate the synthesis and bioactivation of TGF-b.
In contrast to the above mentioned growth factors and cytokines that are
assumed to act from the basolateral site of the epithelial surface and
that seem to stimulate intestinal epithelial restitution through a
common TGF-b-dependent
pathway, various members of the trefoil factor family (TFF Peptide
family) appear to stimulate epithelial restitution in conjunction with
mucin glycoproteins through a TGF-b-independent
mechanism from the apical pole of the epithelium[35,37,38].
In this regard, our group recently also demonstrated that mesalamine
promotes intestinal wound healing in vitro through a TGF-b-independent
mechanism[39]. Recent studies suggest that modulation of
repair mechanisms by trefoil peptides may be mediated by modulation of
the E-cadherin/catenin complex[40,41]. However, a
double-blind, randomized, placebo-controlled study treating 16 patients
with mild-to-moderate left sided ulcerative colitis with enemas
containing human recombinant trefoil factor family-3 did not reveal any
additional benefit above that of adding 5-aminosalicylic acid alone[42].

It has been demonstrated, that keratinocyte growth factor
(KGF) has an important function in wound re-epithelialization[43]
and KGF expression is strikingly increased in surgical specimens from
patients suffering from Crohn’s disease and ulcerative colitis[44].
Furthermore, in an experimental model of colitis, administration of KGF
after but not before induction of colitis significantly ameliorated
tissue damage demonstrating that exogenous KGF might promote IBD[45].
However, clinical studies treating patients with CD or UC with exogenous
KGF have not been performed yet. In addition to their potent effects on epithelial
restitution, a number of regulatory peptide factors act also as potent
modulators of epithelial cell proliferation[17,18,32,46-49].
The most important modulators of intestinal epithelial cell
proliferation include EGF and TGF-b
which both act as potent stimulators of intestinal epithelial
proliferation and TGF-b
which inhibits intestinal epithelial cell proliferation and plays an
important counterbalancing role in the regulation of intestinal
epithelial cell proliferation. TGF-b
is the most potent inhibitor of intestinal epithelial cell proliferation
overriding the stimulatory effects of other stimulatory factors. The
growth stimulating effect of IL-2, FGF peptides, IGF and HGF is rather
moderate compared to the effects of EGF and TGF-b
that stimulate epithelial cell proliferation five- to ten-fold in
several intestinal epithelial cell lines in vitro[33,34,48,50].
Thus, it is not astonishing that in a randomized, double-blind clinical
trial, after a 2-wk treatment period, patients receiving EGF enemas had
a significant lower disease activity score than the control patients[51].
Concerning basic FGF, in a mouse DSS-model of experimental colitis,
rectal administration of human recombinant basic FGF ameliorated the
inflammation score and suppressed TNF-a
gene expression in the colonic tissue[52,53]. Recently, the
important role of Glucagon-like-peptide-2 (GLP-2), which is secreted
from local neuroendocrine epithelial cells and promotes epithelial cell
proliferation via stimulation of enteric neurons[54],
has received more attention. GLP-2 demonstrating ability to ameliorate
murine short bowel syndrome and experimental colitis[55,56]
has consequently lead to clinical studies, evaluating the effect of
GLP-2 in patients with short bowel syndrome.

However, it has also to be considered that cytokines have
pleiotropic activities. For example, FGF also induces stricture
formation in Crohn’s disease[57] and GLP-2 might have
undesired effects in tumorogenesis[58], which might limit
their therapeutic use. In addition, it has to be considered that these
factors and also various non-peptide factors may act in an additive or
even synergistic fashion which may potentiate their single effects.
Notably, in addition to TGF-b
the TGF-b
family member Activin A has been identified to also inhibit epithelial
cell proliferation, thus providing an additional mechanism to
counterbalance the effects of proliferative factors present within the
intestinal mucosa and to inhibit unrestrained cell growth[59,60].

In addition to the potent modulation of intestinal
epithelial wound healing by regulatory peptides, it is increasingly
appreciated that a broad spectrum of non-peptide factors exerts potent
effects on intestinal epithelial cell populations and modulates those
epithelial cell functions that are involved in the healing of intestinal
injury (Table 2).
These non-peptide factors encompass a broad spectrum of unrelated
factors like phospholipids, nutrients (adenine) nucleotides, polyamines,
short chain fatty acids (SCFA), products of the intestinal microflora,
trace elements, pharmacological agents and other factors. Some of these
factors are released by injured or dying mucosal cell populations (e.g.
adenine nucleotides, phospholipids), other reach the intestinal mucosa
via the intestinal lumen or the blood stream. These non peptide
factors may exert growth factor like activities and exert potent effects
on cell growth and differentiation in different cell populations
including fibroblasts, vascular smooth muscle cells, endothelial cells
and keratinocytes[20,35,61-63]. As some of these non-peptide
factors are stable within the gastrointestinal tract despite high
concentrations of acid, bile salts, proteases and microorganisms and as
they exhibit only limited toxicity in vivo, they may serve as
potential future targets to improve the armamentarium for the healing of
mucosal epithelial injury.

Especially phospholipids and polyamines seem to be of
special interest, as these non-peptide factors can be easily added to
the regular diet and their overall content and biological activity can
be modulated by various pharmacological agents[62,64-66].
Lysophosphatidic acid (LPA) is a key intermediate in the early steps of
phospholipid biosynthesis and is rapidly produced and released from
thrombin-activated platelets and growth factor stimulated fibroblasts to
influence target cells by activating a specific 38-40 kDa G-protein
coupled receptor that is expressed in many cells[67]. As a
product of the blood-clotting process, LPA is a normal constituent of
serum, where it is present in an albumin-bound form in physiologically
relevant concentrations[64]. Major sources of LPA in the
vicinity of injured epithelial cells are activated platelets, stimulated
fibroblasts and presumably injured cells that release LPA due to
non-specific phospholipase activation[67]. LPA promotes
platelet aggregation and induces cellular tension and cell surface
fibronectin assembly[68], which are also important events in
wound repair suggesting an important role of LPA in inflammatory
disorders. This was confirmed by our group when we demonstrated, that
LPA not only promotes epithelial wound healing in vitro by a TGF-b-independent
pathway, but also ameliorates experimental colitis in an experimental
model of colitis in rats[61]. Interestingly, also
lysophosphatidylethanolamine and lisofylline, which decreases lipid
peroxidation, significantly reduced the degree of inflammation and
necrosis in an experimental colitis model[69], demonstrating
that the administration of anti-inflammatory lysophospholipids and
suppression of pro-inflammatory lipid metabolites by lisofylline may
provide new approaches to ameliorate intestinal inflammation. This
beneficial effect could also be demonstrated in other diseases since
lisofylline and its analogs reversed autoimmune diabetes in a non-obese
diabetic (NOD) mouse model and thus might act as a potential treatment
for Type 1 diabetes[70,71].

Many studies have reported that bone marrow cells may
have the potential to contribute to the repair of many non-hematopoetic
tissues, including the intestinal epithelial cells. Bone-marrow derived
cells are capable of promoting regeneration of damaged intestinal
epithelial cells[72]. However, the underlying effects are not
fully understood[18]. Nevertheless, bone-marrow
transplantation combined with immune-suppressive therapy improves
epithelial wound healing[73] and recombinant
granulocyte-macrophage colony-stimulating factor have a therapeutic
effect in patients with active Crohn’s disease[74].